Robert W. Boesel

Aging in the rat prostate. Reduction in detectable ventral prostate androgen receptor content.

Abstract Aging in the rat is associated with a reduction in the detectable androgen receptor content of the ventral prostate. The reduction in cytoplasmic receptor content did not appear to be attributable to an aging-associated production of a receptor-inactivating factor or to an aging-associated change in the sedimentation properties of the androgen receptor of young and aged animals. Saturation analysis of cytoplasmic extracts prepared from two different breeds of similar albino rats and a genetically distinct strain of inbred brown rats demonstrated quantitative aging-associated reductions in the androgen-receptor content per cell of the ventral prostate. The reduction in receptor content per cell appeared to increase progressively in magnitude with increasing age. The mean value for the cytoplasmic androgen receptor sites per cell for the oldest animals (mean age 884 days) was only 14% of the mean value for the young mature animals (mean age 185 days) of the same breed. The binding affinities of the detectable androgen receptor of the young mature and aged animals were essentially identical. This observation does not eliminate the possibility that the observed reduction results from an aging-associated production of defective receptor. Evaluation of the total DNA content of the ventral prostate did not provide evidence for an aging-associated selective loss of receptor-containing cells. These data in toto were consistent with the interpretation that aging is associated with a mean reduction in the androgen-receptor content per receptor-containing cell. Both cytoplasmic and nuclear androgen retention were evaluated in vivo. These experiments provided qualitative confirmation of the in vitro saturation analyses as there was a highly significant aging-associated reduction in the amount of androgen specifically bound by these prostatic compartments. Total specific androgen retention by the ventral prostate of aging adults was reduced by 55% relative to young mature animals. This result was nearly identical to that obtained for the same breed and age category of animals when evaluated by in vitro saturation analysis. Preliminary in vitro experiments revealed a diminution in the uptake of androgen receptor by purified nuclei from aged animals relative to purified nuclei from young mature animals. The magnitude of the diminution in nuclear acceptor capacity was insufficient to account for the reduction in nuclear retention of androgen determined in vivo. The data were consistent with the interpretation that the cytoplasmic receptor is the major determinant of nuclear androgen retention in the ventral prostate.

Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate

Saturation protocols were developed for measurement of unoccupied (R) and steroid-occupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2 degrees C) for the synthetic androgen R1881 (17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) and the synthetic progestin R5020 (17alpha,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) respectively was 10.7 +/- 1.4 and 14.3 +/- 3.2 fmoles per mg cytosol protein and the apparent steroid affinity respectively was 9.6 +/- 0.8 and 1.6 +/- 0.4 x 10(8) M-1. Steroid specificity of the unoccupied cytoplasmic R1881 and R5020 binding sites was similar. When R1881 and R5020 were employed as probes of total, R plus RA, cytoplasmic binding components (20-24 hr incubation at 15 degrees C) saturable binding of R5020 was not detectable. Total cytoplasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 +/- 3.3 fmoles per mg cytosol protein and 2.7 +/- 0.6 x 10(7) M-1. R5020 was a poor inhibitor of R1881 binding to total cytoplasmic R1881 binding components.

Crosslinking during the nitration of bovine insulin with tetranitromethane.

Abstract The treatment of bovine insulin with tetranitromethane at pH 8 resulted in the nitration of 2.5 of the four residues of tyrosine in the molecule. Only 0.1 residue of tyrosine was found in the derivative, indicating the modification of 1.4 tyrosine residues by a reaction which did not produce 3-nitrotyrosine. No other amino acid residue besides tyrosine seemed affected by the reaction. Gel chromatography of the derivative in 7 M urea demonstrated the existence of monomers, dimers and larger aggregates of the insulin molecule, an indication of intermolecular covalent crosslinking. The crosslinking also occurred when glycyl- L - tyrosine was treated with tetranitromethane at pH 8.

Androgen regulation of androgen receptor content and distribution in the ventral and dorsolateral prostates of aging AXC rats.

Total androgen receptor content of ventral or dorsolateral prostate of intact, aged (730-740 day old) rats is decreased 50% when compared to intact, young mature (150-170 day old) rats. Treatment with exogenous testosterone increased ventral and dorsolateral prostate androgen receptor content per cell in aged rats to values identical to those of prostates of young mature rats. The increase in prostate receptor content was not attributable to testosterone mediated cellular hypertrophy or hyperplasia. At 24 hr post-orchiectomy ventral prostate cytoplasmic androgen receptors are depleted of endogenous androgen, without any decrease in number of receptors per cell, and nuclear androgen receptors are undetectable. During 30 to 60 min after a single 200 microgram testosterone injection, ventral prostate nuclear receptor content increased to the level of intact control rats without producing any reduction in total cytoplasmic androgen receptor content. Although dorsolateral prostate is devoid of cytoplasmic androgen receptor, the effects of orchiectomy and testosterone treatment upon nuclear androgen receptor are comparable to those seen in ventral prostate. These effects of orchiectomy and testosterone injection upon prostatic receptor content and distribution were identical in prostates of young and aged rats. Our studies show that receptor processing in prostates of young and aged rats does not involve a process by which nuclear receptor is derived by depletion of cytoplasmic receptor. Moreover, our studies of the effect of short-term (48 hr) exogenous testosterone treatment upon androgen receptor content in prostates of aged rats are the first demonstration that androgen receptor content may be enhanced independent of generalized androgen mediated anabolic effects in prostate.

Aging-associated diminished rat prostate androgen receptor content concurrent with decreased androgen dependence

Abstract As inbred AXC rats aged from 137 to 976 days or randomly-bred Sprague-Dawley rats aged from 105 to 610 days, ventral prostate cytoplasmic androgen receptor content decreased 70 and 46%, respectively, and nuclear androgen receptor content decreased 41 and 26%, respectively. Identical evaluations employing dorsolateral prostate failed to provide evidence for a cytoplasmic androgen receptor; however, a 50% decrease in dorsolateral prostate nuclear androgen receptor content was demonstrated in these rats. Prostatic involution subsequent to orchiectomy was employed as a measure of androgen regulation of prostate function in aging rats. Both ventral and dorsolateral prostate evidenced an aging-associated decrease in either the absolute or relative loss in prostate RNA and protein content and wet weight subsequent to orchiectomy. By contrast, the effect of orchiectomy upon cell number was both tissue- and age-dependent. Orchiectomy did not diminish dorsolateral prostate cell number in any of the rats aged 3.5 to 21 months, whereas orchiectomy resulted in profound decreases ventral prostate cell number in rats 3.5 to 16 months of age and did not decrease ventral prostate cell number in rats 21 months of age. The data demonstrate an acquired marked androgen independence for ventral prostate cell number occurring concurrent with an aging-associated diminution in cytoplasmic androgen receptor content and thus suggest a associated diminution in cytoplasmic androgen receptor content and thus suggest a proufound role for cytoplasmic receptor in those processes regulating ventral prostate cell number.

Saturation analysis of the binding of androgens. Antiandrogens and estrogens by the cytoplasmic high affinity androgen receptor of the rat ventral prostate.

Ammonium sulfate fractionation of the cytoplasmic extract obtained from rat ventral prostate yields a protein fraction for which the contribution of nonspecific binding of 5α-dihydrotestosterone to the measured binding of this steroid is markedly reduced. The fractionated binding activity is suitable for utilization in a protocol which permits sensitive, facile and highly reproducible saturation analysis of the high affinity binding of 5α-dihydrotestosterone. The apparent binding constant for this prostatic androgen was 6.9 × 108 l/mol. The number of 5α-dihydrotestosterone binding sites per cell and concentration per 100 mg cytosol protein were 9500 and 8.55 ± 0.36 × 10−12 moles respectively. The apparent binding constants for a number of androgens, antiandrogens and estrogens were determined by evaluation of their ability to inhibit binding of 5α-dihydrotestosterone. Testosterone and all of the common prostatie metabolites of testosterone possessed measurable affinity for the binding component. Our data also demonstrate that the presence of a 3-keto or 17β-hydroxy function on the steroid nucleus is a strong determinant of binding affinity for the prostatic receptor.

Androgen Receptor Content of the Normal and Hyperplastic Canine Prostate

A procedure was developed for measurement of androgen receptors in cytoplasmic extracts of prostates from intact dogs. The protocol utilized exchange saturation analysis at 15 degrees C employing the synthetic androgen R1881 (17beta-hydroxy-17alpha-methylestra-4,9,11-trien-3-one) as the ligand probe and quantitatively detected total cytoplasmic androgen receptor (R(c), androgen-free receptor, and R(c)A, androgen-occupied receptor) present at the initiation of the assay. This protocol was employed in conjunction with a tissue mince saturation analysis procedure (for quantitation of nuclear androgen receptor) to quantitate total androgen receptor content of normal and hyperplastic prostates obtained from young (2.5- or 4.6-yr old) and aged (12.5-yr old) purebred dogs of known birth date. The total cytoplasmic androgen receptor content (picomoles per prostate) of hyperplastic prostates was 4.6-fold greater than that of normal prostates. The total nuclear androgen receptor content of hyperplastic prostates (picomoles per prostate measured in crude nuclear preparations) was either 5.0- (4.6-yr-old dogs) or 7.8-fold (2.5-yr-old dogs) greater than that of normal prostates. However, androgen receptor content per cell was identical for hyperplastic and normal canine prostates, with the exception that nuclear androgen receptor was diminished in prostates from 2.5-yr-old dogs. The cell content per gram dry weight was identical for hyperplastic and normal canine prostates. We conclude that canine prostate hyperplasia is characterized by coordinate proliferation of androgen receptor-positive and androgen receptor-negative cells and is not a consequence of increased accumulation of 5alpha-dihydrotestosterone due to proliferation of androgen receptors per prostate cell.

IDENTIFICATION OF LIMITED CAPACITY ANDROGEN BINDING COMPONENTS IN NUCLEAR AND CYTOPLASMIC FRACTIONS OF CANINE PROSTATE

Nuclear and cytoplasmic androgen receptors were identified in canine prostate preparations. Saturation analysis employing 5α-dihydrotestosterone and tissue from intact dogs demonstrated that crude nuclear pellets contained 85 femtomoles of KCI-extractable and 106 femtomoles of ethanol-extractable receptor sites per 100 μg DNA. The apparent 5α-dihydrotestosterone association constant, 3-3.5 × 108 M−1, and steroid binding specificity of both receptor fractions were identical. 5α-Dihydrotestosterone, 19-nortestosterone, 17β-testosterone, and 5α-androstane-3λ,17β-diol were effective inhibitors of the nuclear binding of radiolabeled 5α-dihydrotestosterone, whereas 5α-androstane-3α,17α-diol, δ4-androstene-3,17-dione, progesterone, estradiol, and cortisol were not. The sedimentation constant of the KCl-extractable nuclear receptor on linear sucrose gradients was 4-5S. Cytoplasmic saturation analysis, employing tissue obtained from dogs 72 hr post-orchiectomy, demonstrated that 5α-dihydrotestosterone and the synt...

A rapid, specific protocol for determination of available androgen receptor sites in unfractionated rat ventral prostate cytosol preparations

A modification of the dextran-coated charcoal technique has been successfully employed for the measurement of androgen receptor binding of 5α-dihydrotestosterone in unfractionated rat ventral prostate cytoplasmic extracts. The addition of a small amount of ethanol to the dextran-coated charcoal solution during the adsorption of unbound ligand greatly facilitated charcoal adsorption of ligand associated with low affinity, high capacity binding components and reduced the contribution of the latter cytoplasmic binding components to less than 10 percent of the measured binding at near saturating concentrations, 10 nM, of 5α-dihydrotestosterone. The assay is facile, sensitive, and highly reproducible and a complete saturation curve can be obtained with as little as 100 mg of ventral prostate. This protocol therefore represents a unique procedure for the quantitation and characterization of the cytoplasmic androgen receptor of rat ventral prostate. The concentration of available cytoplasmic androgen receptor in ventral prostate from young mature (80–120 day old) albino rats, 24 hours post orchidectomy, was 10,300 ± 1780 sites per cell and the apparent binding constant for 5α-dihydrotestosterone was 6.49 ± 0.35 × 108 M−1.

The effect of chronic ingestion of selected pesticides upon rat ventral prostate homeostasis

Abstract The ability of the pesticides carbofuran, chlordane, diazinon, heptachlor, methoxychlor, and parathion to diminish 5α-dihydrotestosterone binding to rat ventral prostate cytoplasmic and nuclear androgen receptors was evaluated by in vitro studies. Only parathion and diazinon consistently inhibited 5α-dihydrotestosterone binding to ventral prostate cytoplasmic or nuclear androgen receptors, the remaining pesticides being relatively ineffective. The data suggested that only parathion and diazinon should be able to affect prostate homeostasis via a mechanism involving altered androgen receptor function. After 90 days of pesticide ingestion, ventral prostate androgen receptor homeostasis was altered in all the rats. Significantly, receptor homeostasis was least affected in diazinon- and parathion-fed rats, whereas ingestion of carbofuran or chlordane produced the most profound changes. Methoxychlor ingestion diminished prostate size and cell content. Carbofuran, chlordane, and parathion, but not heptachlor or diazinon, altered ventral prostate RNA metabolism without affecting cell content. Since pesticide ingestion did not alter mean plasma testosterone concentration, changes in both androgen receptor and prostate homeostasis could be attributed to pesticide action principally occurring in the ventral prostate. The absence of a correlation between pesticide inhibition of 5α-dihydrotestosterone binding to prostate androgen receptors and the pesticides capacity to alter prostate homeostasis demonstrated inhibition studies to be ambiguous predictors of pesticide action in the ventral prostate.